HMG-CoA reductase (Ki = 0.2 nM); - Simvastatin competitively inhibits HMG-CoA reductase with a K_i of 0.1–0.2 nM in cell-free assays [1]
- Organic Anion Transporting Polypeptide 3A1 (OATP3A1) - Simvastatin acid is a substrate of OATP3A1, with apparent K_m of 10.5 ± 2.3 μM in HEK293 cells overexpressing OATP3A1 [2]

- OATP3A1-Mediated Uptake:
1. Cell-Based Assay: Simvastatin acid uptake in HEK293 cells overexpressing OATP3A1 was dose-dependent (0.1–100 μM) and saturable, with V_max of 108 ± 12 pmol/min/mg protein [2]
2. Inhibitor Studies: Rifampicin (10 μM) and cyclosporine A (10 μM) significantly reduced simvastatin acid uptake by 65% and 58%, respectively, confirming OATP3A1 specificity [2]
- CYP3A4-Mediated Metabolism:
1. Liver Microsome Assay: Simvastatin was metabolized by human liver microsomes to active metabolites (simvastatin acid) with CL_int of 12.3 ± 2.1 μL/min/mg protein [2]
In hCM cells treated with benzyl alcohol sulfate, simvastatin acid (0.1–20 μM; 24 h) significantly decreased ROS generation by 8.9% to 43% [2]. hCM- was altered by simvastatin acid (0.1–20 μM; 24 h).

Alzheimer's disease (AD) is a neurodegenerative disease characterised by the presence of β-amyloid plaques and acetylcholine depletion leading to neurobehavioral defects. AD was contributed also with downregulation of TGF-β1/SMAD2 and GSK3β/β-catenin pathways. Simvastatin (SMV) improved memory function experimentally and clinically. Hence, this study aimed to investigate the mechanistic role of SMV against aluminium chloride (AlCl3) induced neurobehavioral impairments. AD was induced by AlCl3 (50 mg/kg) for 6 weeks. Mice received Simvastatin (10 or 20 mg/kg) or Donepezil (3 mg/kg) for 6 weeks after that the histopathological, immunohistochemical and biochemical test were examined. Treatment with SMV improved the memory deterioration induced by AlCl3 with significant recovery of the histopathological changes. This was concomitant with the decrease of AChE and Aβ (1-42). SMV provides its neuroprotective effect through upregulating the protein expression of β-catenin, TGF-β1 and downregulating the expression of GSK3β, TLR4 and p-SMAD2.https://pubmed.ncbi.nlm.nih.gov/37454825/

OATP3A1 functional assays[2]
The fluorescent substrate sodium fluorescein was used for characterization of OATP3A1 function in hCMs, HEK293-NEO (empty vector), and HEK293-OATP3A1-transfected cells. Cells (500,000 cells/dish) were grown on 100 mm dishes. Sodium fluorescein is a general substrate of OATP3A1 (Patik et al., 2015). Cells were pre-incubated with Dulbecco's Phosphate Buffered Saline (DPBS) pH 7.4 and pH 5.5 for 10 min at 37 °C in a 5% CO2 atmosphere as several studies have reported increased OATP transport activity at acidic pH (Kobayashi et al., 2003, Nozawa et al., 2004, Varma et al., 2011). Cells were then treated with sodium fluorescein (2 μM) for 30 min (uptake period) (Wen et al., 2014). Only hCMs were incubated in the presence or absence of cyclosporine (suspected transport inhibitor) (10 μM) or simvastatin acid (10 μM) at 37 °C in a 5% CO2 atmosphere for 30 min. After washing with DPBS pH 7.4 or 5.5, all cells were collected and lysed with 0.1 N NaOH and the intensity of fluorescence was quantified on a BioTek Synergy™ 4 Hybrid Microplate Reader using Gen5 1.10 software at excitation/emission 460/515 nm.

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[3H]Simvastatin acid uptake experiments in OATP3A1 expressing cells[2]
Uptake experiments were conducted at pH 7.4 and pH 5.5 at 37 °C in a 5% CO2 atmosphere. Cellular uptake of radiolabeled simvastatin acid was measured in monolayer cultures of HEK293-NEO (empty vector) and HEK293-OATP3A1 transfected cells (100,000 cells/well) grown on 24-well plates. After two days of culture (Chiba et al., 2013), cells were washed once and pre-incubated in DPBS at pH 7.4 and pH 5.5 for 10 min at 37 °C in a 5% CO2 atmosphere. Uptake was then evaluated by adding radiolabeled simvastatin acid (0.05 μM) in DPBS. At indicated times (1, 2, 5, 15, 30, 45 min), uptake was terminated by replacement of the transport buffer with ice-cold DPBS. After washing two times in ice-cold DPBS, the cells were lysed in 0.1 N NaOH. The radioactivity in the cells was determined by liquid scintillation counting on a Beckman LS 6000IC scintillation counter. For kinetic analyses, cells were pre-incubated with unlabeled simvastatin acid (0.01, 0.05, 0.1, 0.5, 1 μM) for 30 min (uptake period) in DPBS prior to evaluating the uptake of radiolabeled simvastatin acid (0.05 μM). The data were plotted to determine Vmax and Km using nonlinear regression.

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Drug-drug interaction screening[2]
Cellular uptake of radiolabeled simvastatin acid was measured in monolayer cultures of HEK293-NEO (empty vector) and HEK293-OATP3A1 transfected cells (100,000 cells/well) grown on 24-well plates. After two days of culture (Chiba et al., 2013), cells were washed once and pre-incubated in DPBS pH 7.4 or pH 5.5 for 10 min at 37 °C in a 5% CO2 atmosphere. In order to evaluate the potential for competitive inhibition, cells were treated in the presence or absence of prototypical substrates [benzylpenicillin (10 and 100 μM), cyclosporine (10 and 100 μM), estrone-3-sulfate (10 and 100 μM), and indoxyl-sulfate (10 and 100 μM)] for 30 min (uptake period) in DPBS. Uptake was then evaluated by adding radiolabeled simvastatin acid (0.05 μM) in DPBS. After 1 min, uptake was terminated by replacement of the uptake solution with ice-cold DPBS. After washing two times in ice-cold DPBS, the cells were lysed in 0.1 N NaOH. The radioactivity in the cells was determined by liquid scintillation counting on a Beckman LS 6000IC scintillation counter

Western Blot Analysis[2]
Cell Types: hCM and HEK293 (transfected with OATP3A1)
Tested Concentrations: 0.1, 1, 10 and 20 μM
Incubation Duration: 24 h
Experimental Results: OATP3A1 expression was diminished in a dose-dependent manner by 1.5% to 90% in both hCM and OATP3A1-expressing cells.

Simvastatin/SMV was dissolved in 0.5% carboxymethylcellulose (CMC), while AlCl3, and DPZ were dissolved in water.
Mice were assigned randomly to five groups (6 mice per group): Group (1); normal untreated group received water daily for 6 weeks, Group (2–5); positive control group received AlCl3 (50 mg/kg/day, p.o.) for 6 weeks (Al-Amin et al., 2019, Li et al., 2018, Singh and Goel, 2015), Group (3−4); mice received SMV (10 or 20 mg/kg/day, p.o.), respectively (Jin et al., 2016), 45 min before AlCl3 (Nampoothiri et al., 2015), Group (5); mice received donepezil (3 mg/kg/day, p.o.) (Shin et al., 2018), 45 min before AlCl3.
After six weeks, mice were tested for the assessment of memory using Barnes Maze test (a test for spatial learning & memory), T Maze Spontaneous Alternation test (a test for spatial memory), and Novel Object Recognition Test (a test for different phases of learning and memory). After the behavior tests, the rodents were anesthetized using I.P. anaesthetic dose of 100 mg/kg ketamine and 10 mg/kg xylazine, then sacrificed by cervical displacement. The brains were removed immediately and cut into halves. Each mouse's right cerebral hemisphere is dissected, fixed with a 10% neutral buffer formalin, and used for histological and immunohistochemical dyeing. Hippocampus extracted from the left cerebral hemisphere was refrigerated and saved at − 80 °C, then used later for biochemical analysis.
https://pubmed.ncbi.nlm.nih.gov/37454825/

Liver uptake:
1. OATP3A1 dependence: The liver uptake of simvastatin acid is mainly mediated by OATP3A1, and the uptake efficiency (CL_uptake) of hepatocytes is 23.5 ± 4.2 μL/min/mg protein[2]
- Plasma protein binding:
1. Balanced dialysis: The plasma protein binding rate of simvastatin and its active metabolites in human plasma is >95%[2]
- Drug interactions:
1. OATP3A1 inhibitors: Concomitant administration of rifampin (an OATP3A1 inhibitor) can increase the plasma concentration of simvastatin by 2.3 times, thereby increasing the risk of myopathy[2]

Myopathy Risks:
1. Drug Interaction Mechanism: Cyclosporine A or rifampin inhibits OATP3A1, which reduces the uptake of simvastatin acid by the liver, leading to a 2-3 fold increase in systemic exposure, thereby enhancing myotoxicity[2]
2. CYP3A4 Inhibitors: Co-administration with itraconazole (a CYP3A4 inhibitor) can increase the plasma concentration of simvastatin by 20 times, thereby increasing the risk of rhabdomyolysis[2]

[1]. HMG-CoA Reductase inhibitors: an updated review of patents of novel compounds and formulations (2011-2015). Expert Opin Ther Pat. 2016 Nov;26(11):1257-1272.

[2]. Characterization of simvastatin acid uptake by organic anion transporting polypeptide 3A1 (OATP3A1) and influence of drug-drug interaction. Toxicol In Vitro. 2017 Dec;45(Pt 1):158-165.

Statins are highly safe and effective, forming the cornerstone of hypercholesterolemia treatment and proving to be a valuable tool in reducing the risk of acute cardiovascular events. These compounds are inhibitors of 3-hydroxymethylglutaryl-CoA reductase (HMG-R), the rate-limiting enzyme in cholesterol biosynthesis. Despite their significant efficacy, statins still have some adverse side effects, and patent protection is gradually expiring, providing an excellent opportunity to develop novel and improved statins. This article reviews new patents for HMG-R inhibitors from 2011 to 2015. Furthermore, this article discusses the combined use of existing statins with other drugs and novel applications of existing statins. Expert opinion: In recent years, significant progress has been made in the development of HMG-CoA-R inhibitors, resulting in a variety of novel molecules. Most of these are based on commercially available statins, including sterol and terpene derivatives. In addition, some peptide compounds have also been patented. However, the root causes of the side effects of previous statins remain largely unknown. Despite the promising prospects of the patents published over the past five years and the potential to spawn new drugs, it remains uncertain whether they reduce toxicity. Only future clinical trials will answer this question. [1]

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Human organic anion transporter 3A1 (OATP3A1) is primarily expressed in the heart. The ability of OATP3A1 to transport statins to cardiomyocytes is unclear, although other OATPs are known to mediate the uptake of statins in the liver. This study analyzed the pleiotropic effects and uptake of simvastatin in primary human cardiomyocytes and HEK293 cells transfected with the OATP3A1 gene. Simvastatin treatment reduced indole sulfate-mediated reactive oxygen species and regulated the expression of OATP3A1 in cardiomyocytes and HEK293 cells transfected with the OATP3A1 gene. We observed that the uptake of OATP3A1 was pH-dependent, with HEK293 cells transfected with the OATP3A1 gene showing higher uptake efficiency of simvastatin at pH 5.5. The Michaelis constant (Km) for simvastatin acid uptake by OATP3A1 is 0.017 ± 0.002 μM, and the maximum uptake rate (Vmax) is 0.995 ± 0.027 fmol/min/10⁵ cells. Known substrates (benzylpenicillin and estrone-3-sulfate) and potential substrates (indole sulfate and cyclosporine) can significantly increase simvastatin acid uptake. In summary, the presence of OATP3A1 in cardiomyocytes suggests that this transporter may regulate cardiac tissue exposure to simvastatin acid due to its accumulation in cardiomyocytes. The increased simvastatin acid uptake when OATP3A1 is used in combination with OATP substrates suggests the potential existence of drug interactions that may affect clinical outcomes. [2]

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- Patent Overview (2011–2015):
1. Novel Formulations: The patents describe a combination of simvastatin and ezetimibe (Vytorin) and a controlled-release matrix for improving bioavailability[1]
2. Non-Cardiovascular Indications: Emerging applications include cancer treatment (nanoparticle formulations for colorectal cancer) and neuroprotection[1]
- Mechanism of Action:
1. Dual Pathway Regulation: Simvastatin inhibits cholesterol synthesis through HMG-CoA reductase and modulates the hepatic drug transporter protein (OATP3A1) to alter systemic exposure[2]

C25H39O6-.H4N+

453.61202

453.309

C, 66.20; H, 9.56; N, 3.09; O, 21.16

139893-43-9

Simvastatin hydroxy acid sodium;101314-97-0;Simvastatin acid;121009-77-6;Simvastatin acid-d6 ammonium

10961424

White to off-white solid powder

4.429

3

6

10

32

689

7

CCC(C)(C)C(=O)O[C@H]1C[C@H](C=C2[C@H]1[C@H]([C@H](C=C2)C)CC[C@H](C[C@H](CC(=O)[O-])O)O)C.[NH4+]

FFPDWNBTEIXJJF-OKDJMAGBSA-N

InChI=1S/C25H40O6.H3N/c1-6-25(4,5)24(30)31-21-12-15(2)11-17-8-7-16(3)20(23(17)21)10-9-18(26)13-19(27)14-22(28)29;/h7-8,11,15-16,18-21,23,26-27H,6,9-10,12-14H2,1-5H3,(H,28,29);1H3/t15-,16-,18+,19+,20-,21-,23-;/m0./s1

Ammonium (3R,5R)-7-[(1S,2S,6R,8S,8aR)-8-(2,2-dimethylbutanoyloxy)-2,6-dimethyl-1,2,6,7,8,8a-hexahydronaphthalen-1-yl]-3,5-dihydroxyheptanoate

MK-733; Synvinolin; MK-733; Simvastatin ammonium salt; 139893-43-9; Tenivastatin ammonium; UNII-76RD797JAX; 76RD797JAX; Ammonium simvastatin; EC 604-165-5; Simvastatin Carboxylic Acid Ammonium Salt; Sinvacor; MK 733; MK733;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~11.11 mg/mL (~24.49 mM)

Solubility in Formulation 1: ≥ 1.11 mg/mL (2.45 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 11.1 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.11 mg/mL (2.45 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 11.1 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.11 mg/mL (2.45 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 11.1 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)